双歧杆菌质粒聚合酶基因表达系统的构建及其鉴定

目的构建可以在双歧杆菌表达外源性基因的系统。方法PCR扩增双歧杆菌质粒聚合酶基因（BPP）并连接至质粒pBS—T以形成重组质粒pBS—BPP。PCR扩增双歧杆菌内源性阿拉伯糖苷酶的启动子及分泌性信号肽DNA序列（ara）并连接至质粒pBAD—A以形成重组质粒pBAD—ara，然后将增强绿色荧光蛋白（eGFP）基因连接至质粒pBAD—ara以形成重组质粒pBAD—ara—GFP。采用基因重组技术重组含有ara、BPP、eGFP基因序列并可将外源性基因分泌表达于菌体外及锚定表达于细胞壁的质粒pBS—BPP—ara—GFP，激光共聚焦显微镜下观察含有pBS—BPP—ara—GFP质粒及对照质粒的E．coli，验证eGFP定位表达情况。结果所构建的表达系统可以在E．coli中表达eGFP基因。结论通过基因重组方法成功构建了双歧杆菌表达系统，其可将外源基因分泌表达于菌体外。

Construction and identification of Bifidobacterium plasmid polyenzyme expression

Objective To construct and characterize an exogenous gene expression system in Bifidobacterium. Methods The gene coding for Bifidobacterium plasmid polyenzyme （BPP） was amplified and cloned into pBS-T to obtain the plasmid pBS-BPP. The gene coding for ara was amplified and cloned into pBAD-A to obtain the plas- mid pBAD-ara. To characterize the expression system, enhanced green fluorescent protein （eGFP） gene was ampli- fied from the plasmid pcDNAeGFP and cloned into pBAD-ara to obtain the plasmid pBAD-ara-GFP. The recombi- nant plasmid pBS-BPP-ara-GFP was obtained after ligation of plasmid pBS-BPP with plasmid pBAD-ara-GFP. Laser scanning confocal microscope was used to observe the bacteria containing pBS-BPP-ara-GFP and pBS-BPP. Results Green fluorescenece was visualized in the bacteria containing pBS-BPP-ara-GFP, but not in the E. coli containing pBS-BPP. Conclusion The plasmid pBS-BPP-ara-GFP containing BPP and ara are successfully constructed, which are capable of expressing exogenous gene in the E. coli.